Multidimensional detection of aberrant phenotypes in neoplastic cells to be used to monitor minimal disease levels using flow cytometry measurements

Abstract

A method for detecting aberrant phenotypes expressed by neoplastic cells includes the steps of: 1) staining one or more normal / reactive samples and one neoplastic sample with multiple combinations of monoclonal antibodies, 2) measuring fluorescence emissions associated to the stained cells, 3) storing two independent list mode data files of information on light scatter and fluorescence characteristics of each cell, 4) creating new data files by mixing list mode data from the data file containing information the neoplastic sample into the data file containing information on the normal samples, 5) defining corresponding to normal cells and areas corresponding to empty spaces in normal / reactive samples that may be occupied by tumor cells in neoplastic samples, 6) identifying events corresponding to neoplastic cells and events corresponding to normal cells coexisting in a multidimensional space, and 7) establishing the most relevant phenotypic aberrations displayed by the neoplastic cells as compared to their normal counterpart.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of Provisional Application Ser. No. 60 / 451,738 filed Mar. 4, 2003.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]This invention relates to the field of flow cytometry and more particularly to the sequential detection of aberrant patterns of protein expression on neoplastic cells and the identification of minimal numbers of neoplastic cells present among a major population of normal cells from blood, bone marrow, spinal fluid and lymph node samples. The invention enables: 1) the unequivocal identification of the aberrant phenotypes carried by neoplastic cells that allow their sensitive and specific identification, 2) the estimation of their utility for the identification of minimal numbers of neoplastic cells displaying an identically aberrant phenotype in another sample from the same individual obtained simultaneously or subsequently, and 3) the calculation of the distribution of neopla...

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Problems solved by technology

However they could not show the ability of this approach to specifically differentiate between normal and neoplastic cells, coexisting in the same sample.

In U.S. Pat. No. 6,287,791, Terstappen and Chen describe a further refinement of the U.S. Pat. No. 5,047,321, but they did not show any better characterization of the different leukocyte populations.

All the methods described above were able to identify several populations of normal leukocytes present in blood and bone marrow samples and were only identifying selected subpopulations as identified by the specific combination of monoclonal antibodies and nucleic acid dyes used; nevertheless, they were not able to provide an approach for the specific and reproducible identification of neoplastic cells admixtured naturally or artificially with normal cells in a sample.

Moreover, by using these methods it is not possible to easily link and directly compare the information on the amount of light scatter and fluorescence measured for cells contained in a first sample to that of cells containing in a second different sample, especially if they derive from different tissues from the same individual, from different individuals or if they have been measured under different conditions.

The technique described by Ward et al allows the calculation of the absolute counts of leukocytes, such as CD4+ T-cells, but does not provide any specific indication of the exact procedures to be applied for the enumeration of individual subpopulations of blood leukocytes.

However, the need for data interpretation by an expert person with a high amount of knowledge and experience on the patterns of protein expression differentially observed in normal versus neoplastic cells, makes the identification of aberrant phenotypes subjective and difficult to reproduce.

Moreover, many of these aberrant phenotypes are only present in a subset of all leukemic cells present in a given sample and they may change in the same patient, and even in another sample from the same tissue, with time.

This further makes the identification of aberrant phenotypes, apart from being subjective, uncertain, with potentially occurring false negative and positive results.

In addition, current knowledge about the phenotypes of normal cells from blood, bone marrow, spinal fluid and lymph nodes occurring at frequencies of less than 10−4 is very limited; this impacts negatively in the sensitivity of these approaches for detecting minimal numbers of neoplastic cells among a majority of normal blood and bone marrow cells which under the best technical and biological conditions, is currently of between 10−3 (detection of one neoplastic cells among 1000 normal cells) and 10−6 (one neoplastic cell in one million normal cells) depending on the exact lineage, type and maturation stage of the neoplastic cells, the aberrant phenotype used for the identification of the neoplastic cells, and the type of specimen studied.